Project Summary The proteasome is an essential cellular enzyme complex. Its main function is to degrade proteins that have been tagged with ubiquitin. When cells receive a signal, typically a cytokine, the expression of a different isoform of the proteasome, called the immunoproteasome, begins to be produced. The immunoproteasome (iCP) degrades proteins in a similar fashion as the standard proteasome, but more of its products are compatible to be loaded into an MHC-I complex. These MHC-I-peptide complexes are used by cells to initiate the adaptive immune system response by displaying peptides on the cell surface to be recognized by immune cells. The rate and extent of this type of immune system response is critical. For example, when a virus infects cells, it is important the immune system responds rapidly to prevent the virus from replicating too quickly. However, if the immune response is triggered when there is no infection, T-cells can begin to attack and destroy healthy tissue, leading to autoimmune diseases. The inhibition of the immunoproteasome has recently been explored as a potential mechanism to combat autoimmune diseases. The hypothesis is that if less MHC-I compatible peptides can be produced by the iCP, the fewer T-cells will be activated/signaled. However, the opposite is true when a viral infection occurs, when an increase in MHC-I compatible peptides would allow for a rapid immune system response, clearing the virus before it can infect more cells. In this proposal, we will explore how much iCP activity elicits what level of MHC- I expression on a cell. To accomplish this, we will utilize our recently developed iCP-activity probe that can be used in live cells and an antibody to a specific MHC-I-antigen complex using a variety of techniques including confocal microscopy and a plate reader-based assay. While these studies are ongoing, we will also use our activity-based iCP probe to screen for molecules that can affect iCP hydrolysis, leading to a decrease or increase in MHC-I expression. Upon completion of the Aims described here, we will for the first time be able to quantify the relationship between iCP activity and MHC-I expression levels. Additionally, new small molecule inhibitors or stimulators of the iCP will also be discovered and studied. The long-term goal is to use these newly discovered small molecule modulators of iCP activity to affect autoimmune diseases and viral infections.